A T7 RNAP regulatory toolbox for cell-free network engineering and biosensing applications.

T7 RNA polymerase is ubiquitously used in the fields of synthetic biology and biotechnology. Yet the ability to precisely and modularly regulate T7 RNAP remains surprisingly limited. Here, we engineer a T7 RNAP regulatory toolbox consisting of programmable synthetic repressors, activators, and biosensors in a cell-free system. This toolbox enables scalable design of T7 RNAP-based gene regulatory networks and enables rapid, sensitive, and multiplexed detection of diverse biomolecules, including small-molecule drugs, antibodies, and proteins, in a simple one-pot reaction. By integrating a protein design pipeline, we generate biosensors using fully synthetic binders, demonstrating the potential for rapid development of protein-based sensors. We construct a diagnostic cell-free system combining SARS-CoV-2 Spike protein sensing, gene regulatory based amplification, enzymatic amplification, and glucose based detection demonstrating the potential for point-of-care detection with high sensitivity. This work demonstrates a flexible and expandable framework for constructing gene circuits responsive to a wide range of biomolecules and demonstrates the potential for engineering point-of-care cell-free diagnostic assays.